Am J Physiol Cell Physiol 295: C1123–C1132, 2008.
First published September 3, 2008; doi:10.1152/ajpcell.00247.2008.
MDA-MB-231 produces ATP-mediated ICAM-1-dependent facilitation
of the attachment of carcinoma cells to human lymphatic endothelial cells
Yoshiko Kawai, Maki Kaidoh, and Toshio Ohhashi
Department of Physiology, School of Medicine, Shinshu University, Matsumoto, Japan
Submitted 7 May 2008; accepted in final form 28 August 2008
Recently, it has become known that primary tumors influence the microenvironment of tumor tissues before metastasis
(13, 14, 16). However, it is unclear what molecules in the
premetastatic SLN induce a suitable environment for micrometastasis that is related to the attachment of carcinoma cells
to LECs.
Using the human breast carcinoma cell lines MDA-MB-231
and MCF-7, we have attempted to examine the effects of
supernatants cultured with the cell lines on the expression of
adhesion molecules on human LECs and then to investigate
whether the expressed adhesion molecules accelerate the attachment of carcinoma cells to LECs. Thus we have addressed
the possibility that malignant breast carcinoma cells release
chemical substances that make a premetastatic environment
suitable for micrometastasis of carcinoma cells in SLN and its
nearest afferent lymph vessels.
MATERIAL AND METHODS
METASTASIS OF CARCINOMA CELLS
mainly occurs through the
lymphatic system, and the extent of metastasis in lymph nodes
is clinically used as a useful prognostic indication. Recently,
the concept of a sentinel lymph node (SLN), which is the first
node in the lymphatic network draining the primary tumor, has
been proposed with mapping of regional lymph nodes using
radioisotopes or dye. The SLN is the presumptive initial site of
lymphatic micrometastasis of carcinoma cells. The clinical
importance of SLN has been proven in many breast cancer
patients. However, the biological and histological properties of
lymphatic endothelial cells (LECs) in the SLN and its nearest
afferent lymph vessels that interact with micrometastatic carcinoma cells remain unclear. First, we established human LECs
from afferent lymph vessels of SLN in patients with breast
cancers by using trypsin digestion (18, 19).
Cell culture. The isolation and culture of human LECs were
performed using the technique of Kawai et al. (18) with the nearest
afferent lymph vessels of SLN in patients with breast cancer. Human
LECs were maintained in endothelial growth medium (EGM)-2 with
10% fetal bovine serum (FBS) and used at the fifth to seventh passage.
The experimental protocols were approved by the ethical committee
for human studies in the School of Medicine, Shinshu University. All
subjects were informed of the risks and purposes of the studies before
their written consents were obtained.
The human breast adenocarcinoma cell lines MDA-MB-231 and
MCF-7 were purchased from the American Type Culture Collection
(Manassas, VA). The carcinoma cells were maintained in Dulbecco’s
modified Eagle’s medium/Nutrient Mixture F12 Ham (DMEM/F12)
culture medium supplemented with 10% FBS. The LECs were incubated under atmospheric conditions of 5% O2, 5% CO2, and 90% N2
at 37°C, whereas carcinoma cells were incubated under normoxic
conditions of 21% O2, 5% CO2, and 74% N2 at 37°C.
Cytokine and growth factor assays. The concentrations of cytokines and growth factors in the supernatants of the culture media of
MDA-MB-231, MCF-7, or LECs were determined using each specific
ELISA kit. When the supernatants of MDA-MB-231 and MCF-7 were
collected, the carcinoma cells were first plated in DMEM/F12 with
10% FBS, which was replaced the following day with DMEM/F12
with 0% FBS, and cells were collected after overnight culture.
On the other hand, when the supernatant of human LECs was
collected, the LECs were first plated in EGM-2 with 10% FBS, which
was replaced the following day with EBM-2 with 0% FBS (18), and
cells were collected after overnight culture. The collected solution was
centrifuged at 2,000 rpm for 5 min at 4°C and then kept frozen at
⫺20°C for assays of cytokines or growth factors. The concentrations
of tumor necrosis factor (TNF)-␣, transforming growth factor (TGF)␤1, IFN-␥, IL-1␤, IL-6, IL-12, basic FGF (bFGF), PDGF-BB,
Address for reprint requests and other correspondence: T. Ohhashi, Dept. of
Physiology, School of Medicine, Shinshu Univ., Matsumoto, 390-8621, Japan
(e-mail: [email protected]).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
intercellular adhesion molecule-1; sentinel lymph node
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Kawai Y, Kaidoh M, Ohhashi T. MDA-MB-231 produces ATPmediated ICAM-1-dependent facilitation of the attachment of carcinoma cells to human lymphatic endothelial cells. Am J Physiol Cell
Physiol 295: C1123–C1132, 2008. First published September 3, 2008;
doi:10.1152/ajpcell.00247.2008.—We examined the effects of supernatants of culture media of MDA-MB-231 and MCF-7 cells on the
expression of adhesion molecules on human lymphatic endothelial
cells (LECs) and evaluated whether the overexpression of adhesion
molecules facilitated the attachment of carcinoma cells to LECs. The
48-h stimulation of MDA-MB-231, but not MCF-7, supernatant
produced a significant expression of ICAM-1 on human LECs but
little or no expression of E-selectin. Chemical treatment with dialyzed
substances of ⬍1,000 molecular weight (MW) caused a complete
reduction of the supernatant-mediated response. In contrast, pretreatment with heating, digestion with protease, or chemical treatment with
dialyzed substances of ⬍500 MW produced no significant effect on
the supernatant-mediated response. ATP (10⫺7 M) caused overexpression of ICAM-1 on human LECs similar to that produced by the
supernatant of MDA-MB-231. The ATP- and MDA-MB-231 supernatant-mediated responses were significantly reduced by treatment
with 10⫺6 M suramin (a purinergic P2X and P2Y receptor antagonist).
In attachment assays, 10⫺7 M ATP or MDA-MB-231 supernatant
produced a significant increase in the attachment of carcinoma cells to
human LECs. The treatment with 10⫺6 M suramin caused a significant reduction of ATP- and supernatant-mediated facilitation of the
attachment responses. Additional treatment with anti-ICAM-1 antibody also caused a significant reduction of ATP- and supernatantmediated facilitation of the attachment responses. The experimental
findings suggest that MDA-MB-231 may release or leak ATP, which
produces the overexpression of ICAM-1 on human LECs through
activation of purinergic P2X and/or P2Y receptors and then facilitates
ICAM-1-mediated attachment of carcinoma cells to LECs.
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ATP DEVELOPS MICROENVIRONMENT FOR CANCER CELLS
Immunohistochemical studies. Using an indirect immunohistochemical technique, we examined the effects of the supernatants of
culture media of two kinds of carcinoma cells, MDA-MB-231 and
MCF-7, on the expression of adhesion molecules such as E-selectin,
P-selectin, vascular cell adhesion molecule (VCAM)-1, and intercellular adhesion molecule (ICAM)-1 on human LECs. The carcinoma
cells were plated in DMEM/F12 with 10% FBS, which was replaced
the following day with DMEM/F12 with 3% FBS, and then cells were
collected after overnight culture. The collected solution was centrifuged at 2,000 rpm for 5 min at 4°C. To examine the effects of
supernatants on the expression of adhesion molecules on human
LECs, we exchanged the starvation culture medium, EBM-2 with 3%
FBS, with 1 ml of each collected solution, in which the LECs were
stimulated for 4, 18, or 48 h.
Indirect immunohistochemical studies were performed on cultured
LECs seeded on glass slides coated with type I collagen, and then the
cells were fixed with 3.3% formalin or 3% paraformaldehyde in
phosphate-buffered saline solution (PBS) for 20 min at room temperature. The cultured cells were first permeabilized with 0.1% Triton
X-100. Next, the cells were washed three times with PBS and then
Fig. 1. Representative photomicrographs of the effects of starvation culture medium containing 3% fetal bovine serum (FBS) at 0 h of stimulation (A–D),
MDA-MB-231 supernatant at 4 (E–H), 18 (I–L), and 48 h of stimulation (M–P), and MCF-7 supernatant 48 h stimulation of (Q–T) on immunohistochemical
expression of E-selectin, P-selectin, VCAM-1, and ICAM-1 on cultured human lymphatic endothelial cells (LECs). The photomicrographs are merged with
staining of nuclei of the cultured cells. Each marker is 50 ␮m.
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VEGF-A, and VEGF-C were measured commercially (SRL, Tokyo,
Japan). Detection limits were 5 pg/ml for TNF-␣, 0.5 ng/ml for
TGF-␤1, 0.1 U/ml for IFN-␥, 10 pg/ml for IL-1␤, 0.2 pg/ml for IL-6,
7.8 pg/ml for IL-12, 10 pg/ml for bFGF, 31.2 pg/ml for PDGF-BB, 20
pg/ml for VEGF-A, and 109 pg/ml for VEGF-C.
ATP assay. The concentrations of ATP in the supernatants of
culture media of MDA-MB-231 and MCF-7 cells were determined
using the luciferin-luciferase assay based on the Cell Titer-Glo luminescent cell viability assay (Promega, Madison, WI). First, we established a calibration curve for ATP measurement using the same
luciferin-luciferase assay with three kinds of culture media (DMEM/
F12) containing with 10⫺8, 10⫺7, and 10⫺6 M ATP. Next, 100 ␮l of
the supernatant of MDA-MB-231 or MCF-7 cells were collected into
a 96-well plate to which 100 ␮l of luciferin-luciferase solution was
added, and light emission was recorded by a luminometer (Dainippon
Sumitomo Pharma, Osaka, Japan). Thus we calculated the concentrations of ATP in the supernatants of culture media of MDA-MB-231
and MCF-7 cells using the calibration curve for ATP measurement.
The measurements of ATP concentration were done with 10 samples
in each supernatant of MDA-MB-231 and MCF-7, respectively.
ATP DEVELOPS MICROENVIRONMENT FOR CANCER CELLS
Table 1. Summarized data of the measurements of cytokines
and growth factors in MDA-MB-231, MCF-7, or human
LEC culture medium supernatant
TNF-␣, pg/ml
TGF-␤, ng/ml
IFN-␥, IU/ml
IL-1␤, pg/ml
IL-6, pg/ml
IL-12, pg/ml
bFGF, pg/ml
PDGF-BB, pg/ml
VEGF-A, pg/ml
VEGF-C, pg/ml
MCF-7
MDA-MB-231
Human LECs
⬍5
⬍0.5
0.6
18
0.7⫾0.3‡
⬍7.8
⬍10
⬍31.2
312.4⫾6.5†
⬍109‡
⬍5
⬍0.5
0.5
18
611.2⫾102.9*†
⬍7.8
⬍10
⬍31.2
3923.0⫾692.2*†
1108.8⫾142.2*†
⬍5
⬍0.5
0.4
18
0.5⫾0.1
⬍7.8
25
⬍31.2
⬍10
142.2⫾13.4
incubated overnight at 4°C with primary polyclonal human antisera to
E-selectin/CD62E (dilution 10 ␮g/ml), P-selectin/CD62P (dilution 10
␮g/ml), VCAM-1/CD106 (dilution 10 ␮g/ml), and ICAM-1/CD54
(dilution 10 ␮g/ml) (all obtained from R&D Systems, Minneapolis,
MN). After being washed three times in PBS, the cells were incubated
for 1 h at room temperature with 1:100 diluted Alexa Fluor 488
donkey anti-mouse IgG secondary antibody (Invitrogen, Carlsbad,
CA). The nuclei of cultured cells were counterstained and mounted
with ProLong Gold antifade reagent with 4⬘,6-diamidine-2-phenylindole (Molecular Probes, Eugene, OR), examined with a fluorescent
microscope (Leica, Wetzlar, Germany), and photographed.
To evaluate quantitatively the data of the immunohistochemical
studies, in each experiment we examined the cultured cells within
more than three culture dishes and then took photographs of three
images in one culture dish, the image of which usually contained 7–11
culture cells. Thus the total number of the cultured cells examined was
⬃100. For nonspecific staining, Block-ace (Dainippon Sumitomo
Pharma) was substituted for primary antisera as a negative control.
Experimental protocol. To evaluate chemical properties of the
substances released from MDA-MB-231 cells, we studied the effects
of chemically or physically modified supernatants on the immunohistochemical expression of ICAM-1 on human LECs with 48 h of
treatment. In some experiments, the supernatant was boiled at 80°C
for 30 min. The supernatant was treated with protease (pronase E, 1
␮g/ml; Sigma, St. Louis, MO) at 37°C overnight, the reaction of
which was terminated by heating at 80°C for 30 min in some
experiments. In the other experiments, the supernatants were dialyzed
using two kinds of tubing for the dialysis membrane (mol wt 1,000 or
500; Spectrum Medical Industries, Los Angeles, CA). The tubing was
put into a buffer medium [DMEM-F12 (1:1)] for dialysis at 4°C
overnight. The supernatant trapped inside the membrane was then
used for the bioassay. Thus the supernatant contained no chemical
substance ⬍1,000 or ⬍500 in molecular weight.
In the second protocol, to evaluate the pharmacological properties
of the substances released from MDA-MB-231, we investigated the
effects of the supernatant of MDA-MB-231 or ATP (10⫺8 and 10⫺7
M) on the expression of ICAM-1 at 48 h on human LECs in the
absence or presence of suramin (10⫺7 and 10⫺6 M, an antagonist of
P2X and P2Y receptors) (1, 4, 20), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 10⫺7 and 10⫺6 M, a selective adenosine A1 antagonist) (20, 24), or 3,7-dimethyl-1-propargylxanthine (DMPX; 10⫺7 and
10⫺6 M, a selective adenosine A2 antagonist) (20, 24).
To examine quantitatively the data of the immunohistochemical
expression of ICAM-1 on human LECs, we processed high-resolution
digital photomicrographs using the Scion Image analysis program.
Five constant areas of each LEC were outlined on the grayscale image
and processed for the measurement of density. The results are expressed in arbitrary units (mean density/pixel).
In vitro human LEC attachment assay. Human LECs were plated to
form a monolayer on type I collagen-coated 35-mm plates and
incubated to confluence in 5% O2, 5% CO2, and 90% N2 at 37°C. The
Fig. 2. Representative photomicrographs of the effects of 48-h stimulation of IL-6 (A–D), VEGF-A (E–H), or VEGF-C (I–L) on immunohistochemical
expression of E-selectin, P-selectin, VCAM-1, and ICAM-1 on human LECs. Photomicrographs are merged with staining of nuclei of the cultured cells. Each
marker is 50 ␮m.
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Values are concentrations of cytokines and growth factors in the culture
media supernatant of breast cancer cell lines MCF-7 and MDA-MB-231, and
human lymphatic endothelial cells (LECs). *P ⬍ 0.001, significant difference
from MCF-7 supernatant. †P ⬍ 0.001, significant difference from human LEC
supernatant. ‡No significant difference from human LEC supernatant.
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RESULTS
Effects of MDA-MB-231 or MCF-7 supernatant on the
expression of adhesion molecules on human LECs. Figure 1
shows representative photomicrographs of the expression of
adhesion molecules on LECs fixed with 3% paraformaldehyde.
As shown at 0 h, little or no expression of E-selectin, Pselectin, VCAM-1, and ICAM-1 was observed on cultured
human LECs (Fig. 1, A–D). The photomicrographs were
merged with staining of nuclei of the cultured cells. Thus
overnight culture of starvation medium containing 3% FBS
caused little or no expression of adhesion molecules on human
LECs. Similarly, 48-h culture of DMEM/F12 medium containing 3% FBS also produced no significant expression of adhesion molecules on human LECs. In contrast, 4-h stimulation of
the supernatant of MDA-MB-231 cell culture caused a marked
expression of E-selectin on human LECs (Fig. 1E). Thus
almost all cultured LECs were strongly stained by E-selectin
antiserum. Slight staining with ICAM-1 antiserum was, however, observed in human LECs (Fig. 1H). In addition, by
increasing the stimulation time to 18 and 48 h, the immunoreaction of anti-E-selectin was remarkably decreased. On the
other hand, the intensity of the immunoreactivity for ICAM-1
only was significantly increased (Fig. 1, L and P). Thus
immunoreactivities of ICAM-1 were found densely in all
cultured cells examined (⬃100 of 100 cells).
In contrast, 48-h stimulation with MCF-7 supernatant caused
little or no expression of E-selectin, P-selectin, and VCAM-1
on cultured human LECs (Fig. 1, Q–S). Slight staining of
ICAM-1 was, however, observed on the cultured LECs examined (⬃20 of 100 cells; Fig. 1T). The photomicrographs were
also merged with staining of nuclei of the cultured cells.
Measurements of cytokines and growth factors in MDA-MB231 or MCF-7 supernatant. Table 1 shows summarized data of
measurements of cytokines (TNF-␣, TGF-␤1, IFN-␥, IL-1␤,
IL-6, and IL-12) and growth factors (bFGF, PDGF-BB,
VEGF-A, and VEGF-C) in the supernatant of culture medium
of MDA-MB-231, MCF-7, or human LECs. The concentrations of IL-6, VEGF-A, and VEGF-C in the MDA-MB-231
AJP-Cell Physiol • VOL
supernatant were significantly higher than those obtained with
the MCF-7 supernatant (Table 1).
In addition, the concentrations of IL-6, VEGF-A, and
VEGF-C in the MDA-MB-231 supernatant were significantly
higher than those obtained with the LEC supernatant (Table 1).
In contrast, the concentration of VEGF-A only in the MCF-7
supernatant was significantly higher than that obtained with the
LEC supernatant (Table 1). Thus we examined the effects of
48-h treatment with 100 ng/ml IL-6, 100 ng/ml VEGF-A, or
500 ng/ml VEGF-C on the expression of adhesion molecules
on the LECs. The concentrations of IL-6, VEGF-A, and
VEGF-C were chosen to obtain maximal responses of the
cytokine and growth factors on the LECs, taking care of the
cytotoxic effect of these substances. Figure 2 shows representative photomicrographs of the effects of IL-6, VEGF-A, and
VEGF-C on the expression of adhesion molecules on human
LECs. The photomicrographs were merged with staining of
nuclei of the cultured cells. In contrast to the data obtained with
MDA-MB-231 supernatant, some cultured LECs (⬃30 of 100
cells) were slightly stained by ICAM-1 (Fig. 2, D, H, and L).
Little or no expression of E-selectin was observed on the LECs
(Fig. 2, A, E, and I).
Measurement of ATP in MDA-MB-231 or MCF-7 supernatant. To take account of our previous studies showing that ATP
plays pivotal roles in carcinoma cell-mediated regulation of
lymph transport (20, 23), we measured the concentrations of
ATP in the MDA-MB-231 and MCF-7 supernatant using the
calibration curve established with three kinds of culture media
Fig. 3. Calibration curve for ATP measurement obtained with 3 kinds of
culture media containing 10⫺8, 10⫺7, and 10⫺6 M ATP using luciferinluciferase assay. A direct relationship is observed between luminescence
measured with the luciferin-luciferase assay and the concentration of ATP in
the supernatant of culture medium. The abscissa shows negative logarithm of
the concentration of ATP ranging from 10⫺8 to 10⫺6 M. The ordinate denotes
logarithm of luminescence measured with the luciferin-luciferase assay. Filled
circles show mean values of luminescence obtained with the culture media
containing with 10⫺8, 10⫺7, and 10⫺6 M ATP, respectively. The horizontal
and perpendicular bars represent standard errors of the mean (n ⫽ 10). The
open circle indicates the mean value (n ⫽ 10) of ATP concentration obtained
with the MDA-MB-231 supernatant, and the open square is the mean value
(n ⫽ 10) of ATP concentration obtained with the MCF-7 supernatant. †P ⬍
0.001, significantly different from the MDA-MB-231 and the MCF-7 supernatant.
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LECs were kept in serum-starved medium of EBM-2 with 3% FBS.
Selected plates were treated with 10⫺7 M ATP or the supernatant of
culture medium of MDA-MB-231 cells for 48 h. In some experiments,
10⫺6 M suramin was simultaneously added to the plates during 48-h
treatment with 10⫺7 M ATP or the supernatant of MDA-MB-231
cells.
In some experiments, the plates were also treated with 10 ␮g/ml
anti-human ICAM-1 antibody (R&D Systems) for 30 min after 48-h
treatment with the supernatant of MDA-MB-231 or 10⫺7 M ATP.
Breast cancer cells stained with PKH26 fluorescent dye (Sigma) were
then plated at 5 ⫻ 104 cells per plate and incubated for 30 min at
37°C. Unbound cells were removed by aspiration and the plates
washed with DMEM/F12 three times. Attachment was quantitated by
counting cells under ⫻100 magnification using a Leica microscope.
Drugs. All salts were obtained from Wako (Tokyo, Japan). ATP,
suramin, DPCPX, and DMPX were purchased from Sigma. DPCPX
was diluted with ethanol, and DMPX was diluted with DMSO. The
concentrations of ethanol and DMSO did not affect the biological
viability of the culture cells. The concentration of drug was expressed
as the final concentration in the culture plate.
Statistical analysis. All results are means ⫾ SE. Statistical significance was analyzed using Student’s t-test for unpaired observations,
and the value of P ⬍ 0.05 was considered significant.
ATP DEVELOPS MICROENVIRONMENT FOR CANCER CELLS
hand, pretreatment with dialysis membrane ⬍500 mol wt had
no significant effect on 48-h treatment of MDA-MB-231 supernatant-mediated expression of ICAM-1 on human LECs
(Fig. 4A4). Figure 4B4 shows the summarized data [dialysis
⬍500 mol wt, 165.74 ⫾ 3.79 vs. MDA-MB-231 supernatant
(positive control), 179.38 ⫾ 3.49 (n ⫽ 5; not significant)].
Effect of suramin on ATP-mediated expression of ICAM-1
on human LECs. In agreement with the evidence that the
molecular weight of ATP is 551.1 and our previous studies
showing that ATP plays pivotal roles in the carcinoma cellmediated regulation of lymph transport (20, 23), we examined
the effect of ATP on the expression of adhesion molecules on
human LECs. The data are shown in Fig. 5, A1–A3. The 48-h
treatment with 10⫺9, 10⫺8, or 10⫺7 M ATP caused a dosedependent expression of ICAM-1 on human LECs. In particular, 10⫺7 M ATP-mediated expression of ICAM-1 on human LECs was quite similar to the ICAM-1 expression on
the LECs produced by 48-h treatment with MDA-MB-231
supernatant (Fig. 4A1, positive control). In contrast, simultaneous treatment with 10⫺7 or 10⫺6 M suramin caused a
significant reduction of 10⫺7 M ATP-mediated expression
of ICAM-1 on LECs (Fig. 5, A4 and A5). Figure 5B shows
the summarized data [10⫺9 M ATP, 105.33 ⫾ 9.38 (n ⫽ 5);
10⫺8 M ATP, 154.74 ⫾ 6.33 (n ⫽ 5); 10⫺7 M ATP (positive
control), 185.05 ⫾ 6.02 (n ⫽ 5); 10⫺7 M ATP ⫹ 10⫺7 M
suramin, 81.11 ⫾ 15.72 (n ⫽ 5; P ⬍ 0.01 vs. 10⫺7 M ATP);
10⫺7 M ATP ⫹ 10⫺6 M suramin, 85.14 ⫾ 10.72 (n ⫽ 5;
P ⬍ 0.01 vs. 10⫺7 M ATP)].
Fig. 4. A: representative photomicrographs of immunohistochemical expression of ICAM-1 on human
LECs produced by 48-h stimulation of MDA-MB-231
supernatant treated with protease (2), heating (3), and
dialysis of ⬍500 (4) or ⬍1,000 mol wt (5). Micrographs 1 (positive control) and 6 (negative control)
show the representative expression of ICAM-1 produced by 48-h stimulation of MDA-MB-231 supernatant and starvation cultured medium (DMEM/F12 containing 3% FBS), respectively. Each marker is 50 ␮m.
B: summarized data of density measurement of each
photomicrograph using Scion Image analysis. The
number on the abscissa coincides with each photomicrograph in A. The ordinate denotes the normalized
value of the density measurement shown by mean
density/pixel (n ⫽ 5). NS, not significant. **P ⬍ 0.01,
significantly different from the MDA-MB-231 supernatant (positive control)-mediated response.
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containing with 10⫺8, 10⫺7, and 10⫺6 M ATP. The summarized data are shown in Fig. 3. Thus the concentrations of ATP
in MDA-MB-231 and MCF-7 supernatants were estimated to
be 1.03 ⫾ 0.04 ⫻ 10⫺7 and 0.95 ⫾ 0.03 ⫻ 10⫺8 M, respectively. The difference between the MDA-MB-231 and MCF-7
supernatants was statistically significant (P ⬍ 0.001).
Effects of enzymatic digestion with protease, heating, or
dialysis of MDA-MB-231 supernatant on the expression of
adhesion molecules on human LECs. Pretreatment with enzymatic digestion or heating of the MDA-MB-231 supernatant
had no significant effect on the supernatant-mediated expression of ICAM-1 on human LECs [Fig. 4, A1 (positive control)–
A3]. Figure 4, B1 (positive control)–B3, shows the summarized
data [protease, 165.12 ⫾ 2.21 vs. MDA-MB-231 supernatant
(positive control), 179.38 ⫾ 3.49 (n ⫽ 5; not significant);
heated, 162.75 ⫾ 5.36 vs. MDA-MB-231 supernatant (positive
control), 179.38 ⫾ 3.49 (n ⫽ 5; not significant)]. In contrast,
pretreatment with dialysis membrane ⬍1,000 mol wt significantly reduced the expression of ICAM-1 on human LECs
(Fig. 4A5). Figure 4B5 shows the summarized data [dialysis
⬍1,000 mol wt, 92.88 ⫾ 9.82 vs. MDA-MB-231 supernatant
(positive control), 179.38 ⫾ 3.49 (n ⫽ 5; P ⬍ 0.01)]. Thus the
immunohistochemical expression of ICAM-1 on the LECs is
quite similar to the expression of ICAM-1 produced by 48-h
treatment with culture medium of DMEM/F12 with 3% FBS
[Fig. 4A6 (negative control)]. Figure 4B6 shows the summarized data [dialysis ⬍1,000 mol wt, 92.88 ⫾ 9.82 vs. negative
control, 50.62 ⫾ 5.17 (n ⫽ 5; not significant)]. On the other
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Effect of suramin, DPCPX, or DMPX on MDA-MB-231
supernatant-mediated expression of ICAM-1 on human LECs.
The 48-h simultaneous treatment with 10⫺7 or 10⫺6 M suramin
caused a significant reduction of the MDA-MB-231 supernatant-mediated expression of ICAM-1 on human LECs [Fig. 6,
A1 (positive control)–A3]. Figure 6, B1 (positive control)–B3,
shows the summarized data [MDA-MB-231 supernatant (positive control), 157.36 ⫾ 7.88 (n ⫽ 5); MDA-MB-231 supernatant ⫹ 10⫺7 M suramin, 96.31 ⫾ 8.42 (n ⫽ 5; P ⬍ 0.01 vs.
MDA-MB-231 supernatant only); MDA-MB-231 supernatant ⫹ 10⫺6 M suramin, 83.70 ⫾ 4.06 (n ⫽ 5; P ⬍ 0.01 vs.
MDA-MB-231 supernatant only)]. In contrast, 48-h simultaneous treatment with DPCPX (10⫺7 and 10⫺6 M) or DMPX
(10⫺7 and 10⫺6 M) produced no significant effect on the
MDA-MB-231 supernatant-mediated expression of ICAM-1
on human LECs (Fig. 6, A4 –A7). Figure 6, B4 –B7, shows the
summarized data for DPCPX [MDA-MB-231 supernatant
(positive control), 157.36 ⫾ 7.88 (n ⫽ 5); MDA-MB-231
supernatant ⫹ 10⫺7 M DPCPX, 171.14 ⫾ 4.10 (n ⫽ 5; not
significant vs. MDA-MB-231 supernatant only); MDA-MB231 supernatant ⫹ 10⫺6 M DPCPX, 162.86 ⫾ 3.23 (n ⫽ 5; not
significant vs. MDA-MB-231 supernatant only)] and DMPX
[MDA-MB-231 supernatant (positive control), 157.36 ⫾ 7.88
(n ⫽ 5); MDA-MB-231 supernatant ⫹ 10⫺7 M DMPX,
162.77 ⫾ 2.09 (n ⫽ 5; not significant vs. MDA-MB-231
supernatant only); MDA-MB-231 supernatant ⫹ 10⫺6 M
DMPX, 155.92 ⫾ 3.19 (n ⫽ 5; not significant vs. MDA-MB231 supernatant only)].
Attachment assay with 48-h stimulation of ATP or MDAMB-231 supernatant in the presence or absence of suramin. As
shown in Fig. 7, A1 (negative control) and A2, 48-h stimulation
of 10⫺7 M ATP caused a significant increase of the in vitro
attachment of carcinoma cells to human LECs. The increased
attachment of carcinoma cells to LECs was significantly reAJP-Cell Physiol • VOL
duced by simultaneous treatment with 10⫺6 M suramin (Fig.
7A3). The experimental data are summarized in Fig. 7, B1
(negative control)–B3 [DMEM/F12 (negative control), 6.20 ⫾
0.86 (n ⫽ 5); 10⫺7 M ATP (positive control), 17.4 ⫾ 1.21 (n ⫽
5; P ⬍ 0.01 vs. DMEM/F12); 10⫺7 M ATP ⫹ 10⫺6 M
suramin, 10.4 ⫾ 0.93 (n ⫽ 5; P ⬍ 0.05 vs. 10⫺7 M ATP)].
Similar to the stimulation of ATP, 48-h treatment with
MDA-MB-231 supernatant produced significant facilitation of
the in vitro attachment of carcinoma cells to human LECs [Fig.
7A5 (positive control)]. In addition, 48-h simultaneous treatment with 10⫺6 M suramin caused a significant reduction of
the MDA-MB-231 supernatant-mediated increase of the in
vitro attachment of carcinoma cells to LECs (Fig. 7A6). The
experimental data are summarized in Fig. 7, B5 and B6
[DMEM/F12 (negative control), 6.20 ⫾ 0.86 (n ⫽ 5); MDAMB-231 supernatant (positive control), 18.81 ⫾ 0.86 (n ⫽ 5;
P ⬍ 0.01 vs. DMEM/F12); MDA-MB-231 supernatant ⫹ 10⫺6
M suramin, 9.81 ⫾ 0.37 (n ⫽ 5; P ⬍ 0.05 vs. supernatant
only)].
Attachment assay with 48-h stimulation of ATP or MDAMB-231 supernatant in the presence or absence of antiICAM-1 antibody. Next, we examined whether the MDA-MB231 supernatant or ATP mediating the facilitation of the in
vitro attachment of carcinoma cells to human LECs could be
blocked by treatment with 10 ␮g/ml ICAM-1 antibody. The
experimental data are summarized in Fig. 7B4 (n ⫽ 5). As
shown in Fig. 7, A2 (positive control) and A4, the 48-h
stimulation of 10⫺7 M ATP produced a significant increase of
the attachment of carcinoma cells to human LECs. The 10⫺7 M
ATP-mediated increase of the attachment assay was significantly reduced by treatment with anti-ICAM-1 antibody. Figure 7, B2 (positive control) and B4, shows the summarized data
[DMEM/F12 (negative control), 6.78 ⫾ 1.02 (n ⫽ 5); 10⫺7 M
ATP (positive control), 15.7 ⫾ 1.84 (n ⫽ 5; P ⬍ 0.01 vs.
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Fig. 5. A: representative photomicrographs of ATP (1, 10⫺9 M; 2, 10⫺8 M; 3, 10⫺7 M)-mediated expression of ICAM-1 at 48-h stimulation on human LECs
in the absence or presence of suramin (4, 10⫺7 M; 5, 10⫺6 M). Each marker is 50 ␮m. B: summarized data of density measurement of each photomicrograph
using Scion Image analysis. The abscissa and ordinate are as described in Fig. 4. *P ⬍ 0.05 and **P ⬍ 0.01, significantly different from the 10⫺7 M ATP
(positive control)-mediated response, respectively.
ATP DEVELOPS MICROENVIRONMENT FOR CANCER CELLS
C1129
DMEM/F12); 10⫺7 M ATP ⫹ anti-ICAM-1 antibody, 10.06 ⫾
0.81 (n ⫽ 5; P ⬍ 0.05 vs. 10⫺7 M ATP)]. Similar to ATP, 48-h
stimulation of the MDA-MB-231 supernatant also caused a
significant increase of the in vitro attachment of carcinoma
cells to LECs [Fig. 7, A5 (positive control) and B5 (positive
control)]. The increased attachment of carcinoma cells to LECs
was significantly reduced by additional treatment with antiICAM-1 antibody (Fig. 7, A7 and B7) {MDA-MB-231 supernatant (positive control), 18.24 ⫾ 1.89 [n ⫽ 5; P ⬍ 0.01 vs.
DMEM/F12 (negative control)]; MDA-MB-231 supernatant ⫹
anti-ICAM-1 antibody, 7.26 ⫾ 0.85 (n ⫽ 5;P ⬍ 0.05 vs.
supernatant)}.
DISCUSSION
Release of ATP from the human breast cancer cell line
MDA-MB-231. Regional lymph nodes are the most common
and earliest site of metastasis of malignant tumors. The lymph
node acts as a mechanical barrier to prevent the passage of
tumor cells through the node and also acts as a biological
barrier to inhibit tumor growth in the node (5, 6, 7, 12, 22). The
dramatic clinical success of sentinel node navigation surgery
AJP-Cell Physiol • VOL
(26, 27) suggests that the regional lymph node has an effective
filtering function as a mechanical barrier against migrating
cancer cells. On the other hand, it is also known that primary
tumors influence the microenvironment of tumor tissue before
metastasis (14, 16). However, it is unclear what molecules in
the premetastatic regional lymph nodes make a suitable environment for micro-metastasis within the nodes. Thus we have
addressed the possibility that malignant tumor cells release key
chemical substances that can produce a premetastatic environment suitable for micrometastasis of carcinoma cells within
regional lymph nodes.
Our major findings in this study are summarized as follows.
The supernatant of a malignant human breast cancer cell line
with high metastatic ability, MDA-MB-231, caused the selective expression of ICAM-1 on human LECs at 48 h after the
treatment. However, the supernatant of a human breast cancer
cell line with low metastatic ability, MCF-7, produced little or
no expression of ICAM-1 on human LECs. The concentrations
of IL-6, VEGF-A, and VEGF-C in the MDA-MB-231 supernatant were significantly higher than those obtained from the
MCF-7 or the human LEC supernatant. However, ⬃25- to
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Fig. 6. A: representative photomicrographs of MDA-MB-231 supernatant-mediated expression of ICAM-1 at 48-h stimulation on human LECs in the absence
(1, positive control) or presence of suramin (2, 10⫺7 M; 3, 10⫺6 M), DPCPX (4, 10⫺7 M; 5, 10⫺6 M), or DMPX (6, 10⫺7 M; 7, 10⫺6 M). Each marker is 50
␮m. B: summarized data of density measurement of each photomicrograph using Scion Image analysis. The abscissa and ordinate are as described in Fig. 4.
**P ⬍ 0.01, significantly different from the MDA-MB-231 supernatant (positive control)-mediated response.
C1130
ATP DEVELOPS MICROENVIRONMENT FOR CANCER CELLS
500-fold higher concentrations of the cytokine and growth
factors caused a slight expression of ICAM-1 on human LECs,
being not similar to the MDA-MB-231 supernatant-mediated
expression of ICAM-1 on LECs. Chemical treatment with
dialyzed substances of ⬍1,000 mol wt caused a complete
reduction of the MDA-MB-231 supernatant-mediated expression of ICAM-1 on human LECs. In contrast, pretreatment
with heating, enzymatic digestion of the MDA-MB-231 supernatant with protease, or chemical treatment with dialyzed
substances of ⬍500 mol wt produced no significant effect on
the supernatant-mediated expression of ICAM-1 on human
LECs. These findings suggest that the human breast cancer cell
line MDA-MB-231 may release nonpeptide substance(s) of
⬎500 and ⬍1,000 mol wt.
On the other hand, the concentration of ATP in the MDAMB-231 supernatant may be expected to be about 1.03 ⫾ 0.04
⫻10⫺7 M. In contrast, the concentration of ATP in the MCF-7
supernatant (0.95 ⫾ 0.03 ⫻ 10⫺8 M) was significantly lower
than that obtained with the MDA-MB-231 supernatant. In
addition, taking account of our previous studies showing that
ATP plays pivotal roles in carcinoma cell-mediated regulation
of lymph transport (20, 23), we examined the effects of ATP on
the expression molecules on human LECs and found that 10⫺7
M ATP caused the same expression of ICAM-1 on human
LECs as that produced by the MDA-MB-231 supernatant. Pretreatment with 10⫺7 and 10⫺6 M suramin (a P2X and P2Y
receptor antagonist) produced a significant reduction of ATP- and
MDA-MB-231 supernatant-mediated expression of ICAM-1 on
LECs. The concentration of suramin is known to selectively block
P2X and P2Y receptors (20). In contrast, 10⫺7 and 10⫺6 M
DPCPX (a selective adenosine A1 antagonist) or 10⫺7 and 10⫺6
M DMPX (a selective adenosine A2 antagonist) had no significant
effect on the MDA-MB-231 supernatant-mediated expression of
ICAM-1 on human LECs. Therefore, we concluded that a malignant human breast cancer cell line with high metastatic ability,
MDA-MB-231, but not MCF-7 with low metastatic ability, might
release or leak ATP, which induces the selective expression of
AJP-Cell Physiol • VOL
ICAM-1 on human LECs through the activation of purinergic
P2X and/or P2Y receptors on LECs. This conclusion is strongly
compatible with the present experimental finding that cytokines
and growth factors such as IL-6, VEGF-A, and VEGF-C had no
or little effect on the expression of ICAM-1 on human LECs. This
conclusion also agreed with evidence that the molecular weight of
ATP is 551.1, between 500 and 1,000 mol wt.
However, it remains unclear whether purinergic P2X and/or
P2Y receptors are expressed on MDA-MB-231 breast cancer
cells and then whether activation of the purinergic P2X and/or
P2Y receptors on MDA-MB-231 would be involved in building up the premetastatic environment within regional lymph
nodes. Thus further investigation is needed to evaluate pivotal
roles in activation of the purinergic P2X and/or P2Y receptors
on MDA-MB-231 breast cancer cells for development of the
microenvironment within the lymph nodes.
In addition, this conclusion may be strongly supported by
our previous physiological studies that a malignant melanoma
cell line, B16-BL6, may release nonpeptide substance(s) of
⬍1,000 mol wt, resulting in significant cessation of lymphatic
pump activity via the production and release of endogenous
nitric oxide from lymphatic endothelial cells (23). ATP also
caused significant dilation with the cessation of lymphatic
pump activity. ATP-induced dilation and inhibition of pump
activity of isolated rat lymph vessels are endothelium dependent. Thus ATP-mediated inhibitory responses may be, in part,
released to produce endogenous nitric oxide in lymphatic
endothelium (20). It is reasonable to hypothesize that a high
concentration of ATP released or leaked from malignant primary tumors, such as MDA-MB-231 and B16-BL6, diffuses
the interstitial space, penetrates the lymph capillaries, modulates active lymph transport mechanisms, and then produces a
premetastatic environment suitable for micrometastasis of carcinoma cells within regional lymph node(s). Thus ATP causes
dilation of lymph vessels and reduction of lymphatic pump
activity, which may lead to decreased lymph flow, resulting in
edema of the tumor tissues. Microenvironmental edema in the
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Fig. 7. A: representative dark-field photomicrographs of the in vitro attachment assay of the effects of 48-h stimulation of ATP (2, positive control) or
MDA-MB-231 supernatant (5, positive control) in the absence or presence of 10⫺6 M suramin (3 and 6, respectively) or anti-ICAM-1 antibody (4 and 7,
respectively). B: summarized data of the attachment assay normalized using the number of adherent carcinoma cells per field (⫻100). *P ⬍ 0.05, significantly
different from the stimulation of ATP (positive control) or MDA-MB-231 supernatant (positive control). **P ⬍ 0.01, significantly different from the negative
control (A1 and B1, the stimulation of starvation culture medium only) or the treatment with anti-ICAM-1 antibody.
ATP DEVELOPS MICROENVIRONMENT FOR CANCER CELLS
AJP-Cell Physiol • VOL
(15). On the other hand, studies of leukocyte-endothelial cell
adhesion tumor microvessels have revealed diminished adhesive interactions under both basal and cytokine-stimulated
conditions (28). This observation is consistent with immunohistochemical (21) and cytofluorimetric (11) studies that predicted the reduced endothelial ICAM-1 expression in tumor
microvessels. It has been suggested that the proposed downregulation of endothelial ICAM-1 facilitates tumor progression
by allowing tumor cells to avoid immunosurveillance by circulating lymphocytes. There are, however, several other immunohistochemical studies of tumor vasculature that invoke
the enhanced expression of endothelial ICAM-1, resembling an
inflammatory phenotype, in non-small cell lung carcinoma (25)
and breast cancer (8). The expression of adhesion molecules on
human LECs remains unclear.
In the present experiments, MDA-MB-231 may have released or leaked ATP, which can produce the overexpression
of ICAM-1 on human LECs and then facilitate ICAM-1mediated attachment of carcinoma cells to LECs located in the
nearest SLN of patients of breast cancer. However, to evaluate
quantitatively ATP-mediated upregulation of ICAM-1 adhesion molecule on human LECs, additional analyses of protein
expression using Western blotting are needed in the future.
Thus there exists no information, except for the present study,
regarding the effects of ATP on human LECs nearest and/or
within SLN(s) with special reference to the expression of
adhesion molecules and interaction with carcinoma cells such
as the development of a premetastatic microenvironment and
micrometastasis of carcinoma cells. Therefore, this study may
be the first to suggest that ATP released and/or leaked from
malignant carcinoma cells with high metastatic ability may
play crucial roles in the establishment of a premetastatic
environment within the regional lymph node(s) and the development of micrometastasis of carcinoma cells with high metastatic ability.
ACKNOWLEDGMENTS
We thank Drs. K. Hosaka, R. Mizuno, and F. Ikomi, as well as Y.
Yokoyama, of the School of Medicine, Shinshu University, for valuable
support in the promotion of this research project.
GRANTS
This study was supported financially, in part, by Japanese Ministry of
Education, Science, Sports and Culture Grants-in-Aid for Scientific Research
17591873 and 19209044 and by the Intelligent Surgical Instruments Project of
the Japanese Ministry of Economy, Trade and Industry (2007).
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